The invention relates to optimized adhesins and nanoparticles to which said adhesins are bound. The invention furthermore relates to providing said nanoparticles by way of in vivo contrast agents, in particular for the diagnosis of bowel cancer.
For many diseases, a diagnosis which is both as early as possible and meaningful is of crucial importance for choosing as well as coordinating and performing the necessary medical measures. This is especially true for many kinds of tumors, for the determination and therapy of which (including possible sections) it is essential to discriminate between healthy and carcinogenic tissues. Accordingly, the recovery or even survival of a patient crucially depends on, whether and to what extent the physician in charge of treatment and/or surgery is able to distinguish different types of tissues.
To improve diagnosis and medical measures, contrast agents have been developed in the past which can help with making visible functions and structures in the body by imaging processes. These processes are used inter alia for specifically detecting cancer-associated cellular alterations.
WO 2007/057182 A3, for example, discloses fluorescent nanoparticles comprising at least three structures, namely an inorganic core which is coated with a passivation layer which in turn carries specific ligands. These ligands allow said nanoparticle to specifically bind to a target. Said target may especially be specific surface molecules of the target cells, for example tumor-associated antigens.
The nanoparticles with core and surrounding passivation layer, which are disclosed by this printed publication, have a small hydrodynamic diameter which preferably is between 5 and 15 nm. Nanoparticles of this size can be illuminated via the kidneys and therefore do not, or at most only in tolerable amounts, accumulate in the body. This is one of the important requirements for using nanoparticles for medical purposes.
Another requirement is the high specificity of the ligands bound to the nanoparticles recognizing the selected target structure in the body. Only high-specific recognition allows detection which is the main purpose of the medical use of a contrast agent. These highly specific ligands must at the same time be so small that they do not impede good distribution and penetration of the nanoparticles. Frequently employed specific ligands such as antibodies or Fab fragments are usually too large in order to meet this requirement.
The number of new cases of bowel cancer in industrialized countries has risen significantly over the last 30 years. With an annual incidence of new cases of 30-35 per 100 000 inhabitants, bowel cancer is one of the most common malignancies in central Europe and is responsible for about 15% of all cancer fatalities. The worldwide incidence is estimated to be a million new cases per year. Men are slightly more affected than women, especially with rectal carcinomas (sex ratio 60:40).
In Germany, bowel cancer is the second most common form of cancer, both with new cases and with cancer fatalities in men and women, with more than 20 000 people having died in 2005. The “Gesellschaft der epidemiologischen Krebsregister [Society of epidemiological cancer registers]” even assumes that there are nearly 30 000 fatalities per year.
Bowel cancer refers to any malignant tumors of the intestines. They may be carcinoids, especially in the appendix and the small intestine, leiomyosarcomas and gastrointestinal stromal tumors (GISTs) which derive from the smooth muscle and/or the connective tissue of the intestinal mucosa. However, these diseases are rather rare and represent only a small proportion of all bowel cancer cases. The by far largest proportion of all bowel cancer cases (more than 95%) is represented by adenocarcinoma of the cecum, colon or rectum, for which the generic term colorectal cancer is also used.
Bowel cancer, especially colorectal carcinomas, vary rarely cause symptoms initially and nearly always develop from initially benign intestinal polyps. The chances of curing by surgery and subsequent chemotherapy with a 5-year rate of survival of, on average, from 40 to 60% crucially depends on the stage at which the bowel cancer is diagnosed. Preventive care and also early diagnosis are therefore of particular importance. The latter would be possible by way of early detection of bowel cancer cells, in particular CRCs (colorectal cancer cells).
Detection of CRCs requires a target which is specific to said cells and, if possible, is overexpressed in the tumor. Members of the CEACAM family (CEA-related cell adhesion molecule; CEA=carcinoembryonic antigen) are reported as having upregulated expression in CRC cells. CEACAM family members are therefore particularly suitable as targets for CRC detection. They include especially CEA (CEACAM5) and NCA (CEACAM6).
The CEACAM family is a member of the Ig superfamily. Each family member is highly glycosylated and consists of an N-terminal Ig variable-like domain, downstream of which there are up to 6 IgC2 domains. CEACAM1, CEACAM3 and CEACAM4 are inserted via a carboxy terminal transmembrane domain and cytoplasmic domain in the cell membrane, whereas CEA (CEACAM5), CEACAM6 (NCA), CEACAM7 and CEACAM8 are anchored via glycosyiphosphatidylinositol (GPI) on the membrane. The N-terminal domain within this group has more than 90% similarity at the amino acid level.
CEA and NCA are present in the cylindrical epithelium and in the goblet cells in colon tissue. There they are located on the apical surface of mature enterocytes, that is in the glycocalyx/microvillus region. CEACAM act as intercellular adhesion molecules. This strictly apical localization of CEACAM in normal colon epithelial cells has been abandoned in adenocarcinoma cells—the proteins are thus expressed on the entire cell surface. Cellular organization and cellular polarity has been abolished in CRC cells, and thus, for example, CEA and/or NCA can be found located on the entire cell surface. CEA may be released, thereby entering the bloodstream and thus being detected as serum tumor marker.
Ligands which have been reported for these receptors are inter alia adhesins of the Dr family of E. coli bacteria (Afa/Dr adhesins, DrCEA subfamily). These adhesins are located on the bacterial surface, partly organized in the fimbriae, of diffusely adhering E. coli (DAEC) strains and mediate adhesion thereof to epithelial cells. Members of this family are AfaE-I, AfaE-III, AfaE-V, DraE and DaaE, for example.
The structural assembly genes coding for Afa/Dr adhesins have a similar organization. They consist of operons comprising at least 5 genes (A to E). Genes A to D here encode accessory genes, with gene D encoding an invasin. Gene E encodes the actual adhesin.
The gene clusters have highly conserved regions, for example the genes afaA, afaB, afaC, afaD and afaF, which have regulatory or chaperone function, for example. The structural, AfaE-encoding gene is very heterogeneous, resulting in antigenically different adhesins being produced.
E. coli bacteria that express these members of the Afa/Dr family of adhesins adhere to CHO cells expressing CEA (Berger et al., Molecular Microbiology, (2004) 52(4), pp. 963-983, “Differential recognition of members of the carcinoembryonic antigen family by Afa/Dr adhesins of diffusely adhering Escherichia coli (Afa/Dr DAEC)”). Furthermore, adhesins are also described in Alain L. Servin (Clinical Microbiology Reviews (April 2005) 18(2), pp. 264-292, “Pathogenesis of Afa/Dr Diffusely Adhering Escherichia coli”).
However, these known adhesins are still unsatisfactory with regard to their affinity for CEA. It is therefore an object of the present invention to provide adhesins or adhesin fragments (“adhesin constructs”) which have high affinity for CRC cells. For this purpose, they should have an improved affinity for CEA over the known wild-type adhesins, if possible.